Utilisation of Oxford Nanopore sequencing to generate six complete gastropod mitochondrial genomes as part of a biodiversity curriculum

High-throughput sequencing has enabled genome skimming approaches to produce complete mitochondrial genomes (mitogenomes) for species identification and phylogenomics purposes. In particular, the portable sequencing device from Oxford Nanopore Technologies (ONT) has the potential to facilitate hands-on training from sampling to sequencing and interpretation of mitogenomes. In this study, we present the results from sampling and sequencing of six gastropod mitogenomes (Aplysia argus, Cellana orientalis, Cellana toreuma, Conus ebraeus, Conus miles and Tylothais aculeata) from a graduate level biodiversity course. The students were able to produce mitogenomes from sampling to annotation using existing protocols and programs. Approximately 4 Gb of sequence was produced from 16 Flongle and one MinION flow cells, averaging 235 Mb and N50 = 4.4 kb per flow cell. Five of the six 14.1–18 kb mitogenomes were circlised containing all 13 core protein coding genes. Additional Illumina sequencing revealed that the ONT assemblies spanned over highly AT rich sequences in the control region that were otherwise missing in Illumina-assembled mitogenomes, but still contained a base error of one every 70.8–346.7 bp under the fast mode basecalling with the majority occurring at homopolymer regions. Our findings suggest that the portable MinION device can be used to rapidly produce low-cost mitogenomes onsite and tailored to genomics-based training in biodiversity research.


Field sampling
Prior to the start of a graduate class taught at Taiwan International Graduate Program for Biodiversity 1 , one sample (DJ) was collected and sequenced in Ruifang (25°07'17.8"N, 121°49'19.9"E), Taiwan to test the protocols laid out in this study. All materials were used for this sample, hence no voucher was kept. The students went on and collected five Gastropoda samples from the rocky shores in Da Bai Sha (Green Island, Taiwan, 22.639° N, 121.493° S; WGS84, see Supplementary Figure  S1) during low tide on 22nd March 2021. Specimens were either collected by hand or with tweezers. The specimens were placed in 2-5 L transparent bottles with sea water and transported back to the lab at the Green Island Marine Research Station, Marine Science Center, Academia Sinica, Taiwan. Here, they were separated by taxon and kept in 10-20 L aquaria in aerated seawater. The specimens were not fed prior post-sampling processing. After collecting explants/tissue samples for DNA extraction, the remaining parts of the specimens were fixed in 95% ethanol and brought to Academia Sinica Museum of Natural History (sample IDs ASIZM0001713, ASIZM0001714, ASIZM0001715, ASIZM0001718, ASIZM0001719). Each specimen was carefully removed from their aquarium tanks and placed on roughly 20 x 20 aluminum foil pieces. We used sterilized scissors or razor blades to dissect four 25 mg muscle tissues from each specimen of either the subepithelial tissue, muscular foot or heart.

Morphological description of gastropods
Aplysia is a genus of sea slugs under the order Anaspidea and family Aplysiidae. Sea slug species have highly reduced internal shells and the body of several Aplysia species is characterized by ring-like spots over the head and parapodia. Given this, species identification can be challenging if done morphologically, due to the presence of color and marking polymorphism 2 . In particular, A. argus resembles A. oculifera and A. dactylomela, although the latter is not regarded as occurring in the Indo-Pacific region anymore and the mentioned species can be recognized by their internal shell morphology 2 .
Cellana is a limpet (superfamily Patelloida) genus belonging to the family Nacellidae, known for having a singular flattened shell and for being grazers which feed on algae as food 3,4 . Cellana is widespread in the Indo-Pacific area 5 and its species were usually recognized by shell morphology, although its high variability made it complex and now usually radular and body characteristics are used instead 4 . As a proof of the complexity of the characters used for species delimitation in this genus, Ce. orientalis has a very controversial taxonomy and was once regarded as a subspecies of Ce. radiata 6 . The latter is regarded as a species complex 4 . As a possible morphological marker, Powell 7 reports "very distinct radial folds that underlie the normal radial sculpture" in Ce. orientalis (although it was still regarded as a subspecies of Ce. radiata at that time). Regarding Ce. toreuma, it is widely distributed in East Asia. It presents wide variation in sculptural and color characters, although it presents a depressed conical shape and the aperture is usually oval. It has been present in Asia at least since the Middle Pleistocene 8 .
The genus Conus is a predatory clade, with more than 750 described species 9 . It has thick coiled shell with the whorls enrolled upon themselves and short shell spire. The aperture is narrow and elongated with parallel margins and they also have a needle-like modified radula with a venom gland to attack and paralyze the prey and then engulf it [9][10][11] . Both the species we sampled feed on polychaetes 10 . From what concerns Co. ebraeus, it superficially resembles Co. judaeus due to the presence of black botches on the white shell, although their radula and their feeding preferences (Co. ebraeus feeds on Eunicidae, while Co. judaeus on syllids) are different 12 . Co. miles, instead, share some very vague similarity with Co. capitaneus but it has brownish lines around the whorls and spire 10 .
Tylothais aculeata is a murex (family Muricidae) snail, which preys on other intertidal invertebrates 13,14 . The shell is coiled and has shell spines 14 . It was recently erected as a standalone genus, due to the fact the previous genus (Thalessa) is now regarded as a junior synonym of Volema 14 . It was regarded as a Mancinella species in Taiwan faunal checklist 15 .

Design of student bioinformatic class
Prior to the analysis class, a questionnaire was given to the students to assess their experiences in genome skimming and familiarity with Linux. As approximately half of students had limited or no experiences with Linux, the whole exercise was designed to run under three hours and was conducted by pairs of students with at least one student having some experiences with Linux. The whole exercise is available at 16 .
As MitoZ required more time to run, the finished results were prerun and available to students who have completed all the exercise for further interpretation of the annotated results.

Supplementary Figures
Supplementary Figure S1  Supplementary Figure S5 -Aplysiidae phylogeny. The topology of the phylogeny was inferred using a coalescence of 13 mitogenome gene phylogenies from ASTRAL 17 . Red colour denotes species sequenced in this study. Figure S6 -Patellogastropoda phylogeny. The topology of the phylogeny was inferred using a coalescence of 13 mitogenome gene phylogenies from ASTRAL 17 . Red colour denotes species sequenced in this study. Figure S7 -Conidae phylogeny. The topology of the phylogeny was inferred using a coalescence of 13 mitogenome gene phylogenies from ASTRAL 17 . Red colour denotes species sequenced in this study. Figure S8 -Muricidae phylogeny. The topology of the phylogeny was inferred using a coalescence of 13 mitogenome gene phylogenies from ASTRAL 17 . Red colour denotes species sequenced in this study.